Biosensors are defined as fluidic systems with cavities and/or channels, which are used to measure the molecular interactions of diffusing biomolecules with other at the surfaces of the biosensors immobilized molecules. A majority of the current biosensor developments are intended for bioengineering and biotechnology applications. In the scope of this invention, biosensors are used to measure biomolecular interactions for in vitro diagnostic applications.
Swiss patent application CH 01824/09 discloses biosensors for the detection of biomolecular interactions. The biosensors were described for a use with a confocal microscope. However, confocal microscope reading is difficult to automate, leading to long measurement times.
Current technologies for the detection of biomolecular interactions can be divided in two categories: (a) the labeled techniques and (b) the label-free techniques.
Among the labeled techniques, the widely used methods are fluorescence, colorimetry, radioactivity, phosphorescence, bioluminescence and chemiluminescence. Functionalized particles such as nanoparticles or magnetic beads can also be considered as labeling techniques. Their advantages are the sensitivity in comparison to label-free methods and the molecular specificity due to specific labeling.
Fluorescence microscopy allows to measure the presence and the concentration of biomolecules specifically labeled with a fluorescent molecule called a fluophore. The specimen is illuminated with light of a specific wavelength, which brings it to an excited state, leading to an emission of light at a longer wavelength. The emission is measured by a detector, which allows quantifying the number of fluophores in the measurement volume.
Fluorescence correlation spectroscopy (FCS), as a known representative of single molecule detection techniques, allows to access, across the fluctuation analysis of fluorescently labeled single biomolecules, static and dynamic molecular parameters, such as the mean number of molecules, their diffusion behavior and kinetic binding constants. This single molecule detection tool enables to measure the specificity of the biomolecule interaction, without being influenced by the presence of the fluorescent molecules outside the detection volume.
In close relation to FCS several other techniques, known as Photon Counting Histogram (PCH), Fluorescence Intensitiy Distribution Analysis (FIDA) or Fluorescence Lifetime spectroscopy (FLS), use the intrinsic fluorophore mediated properties of single biomolecules for measuring the chemical binding constants, concentration or number of molecules, diffusion properties, etc. All these techniques are substantially compatible with the disclosed invention.
Nanoparticle-based microscopy is an emerging technique allowing detecting the presence of functionalized nanoparticles that can be attached on biomolecules of interest. This technique has several advantages over fluophores such as chemical stability and no photobleaching.
Among the label-free techniques, the widely used are electrochemical biosensors, referring to amperometric, capacitive, conductometric or impedimetric sensors, which have the advantage of being rapid and inexpensive. They measure the change in electrical properties of electrode structures as biomolecules become entrapped or immobilized onto or near the electrode. However, all these concepts lack molecular specific contrast, sensitivity and reliability.
Surface plasmon resonance (SPR) is also a label-free optical technique for monitoring biomolecular interactions occurring in very close vicinity of a transducer gold surface, and has lead to great potential for real-time studying surface-confined affinity interactions without rinsing out unreacted or excess reactants in sample solutions. However, this method is limited to ensemble measurements, meaning that it is not single-molecule sensitive.
The other important technologies for biomolecular diagnostics are Western and Northern blots, protein electrophoresis and polymerase chain reaction (PCR). However, these methods require highly concentrated analytes.